title: Using the recognition code to swap homeodomain target specificity in cell culture.
abstract: The homeodomain (HD) is a 60 amino acid-long DNA-binding domain. A large fraction of HDs binds with high affinity sequences containing the 5'-TAAT-3' core motif. However, NK-2 class HDs recognizes sequences containing the 5'-CAAG-3' core motif. By using a cell transfection approach, here we show that modification of residues located in the N-terminal arm (at positions 6, 7 and 8) and in the recognition helix (at position 54) is enough to swap the "in vivo" binding specificity of TTF-1 HD (which is a member of the NK-2 class HD) from 5'-CAAG-3' to 5'-TAAT-3'-containing targets. The role of residue at position 54 is also supported by data obtained with the HD of the Drosophila engrailed protein. These data support the notion that DNA-binding specificity "in vivo" is dictated by few critical residues.
authors.affiliation: Dipartimento di Scienze e Tecnologie Biomediche, Università di Udine, Piazzale Kolbe 1-33100 Udine, Italy.
authors.forename: Cinzia
authors.initials: C
authors.lastname: Puppin
authors.forename: Dora
authors.initials: D
authors.lastname: Fabbro
authors.forename: Lucia
authors.initials: L
authors.lastname: Pellizzari
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Damante
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001681:Biological Assay; D018929:Cell Culture Techniques / Q000379:methods*; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D017930:Genes, Reporter / Q000235:genetics; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D000596:Amino Acids; D029721:Drosophila Proteins; C063459:En protein, Drosophila; D018398:Homeodomain Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors